1. Technical Field of the Invention
The present invention relates to the synthesis of oligonucleotide analogs. More particularly, the invention relates to a novel polymeric disc, wafer or other similarly shaped resin (e.g., planar and non-planar) and a method for its use in solid-phase synthesis.
The present invention permits the rapid production of oligonucleotide analogs, i.e., numerous oligonucleotides differing from one another by only a single base or a small number of bases. The synthesis of analogs according to the present invention can take place at a rapid rate while assuring that the reagents necessary to synthesize the analogs undergo quantitatively complete reactions so as to minimize undesirable side-reaction products which could result in the production of "deletion peptides" or "deletion sequences."
Within recent years, oligonucleotides have been used in crystallographis and biochemical studies of DNA and RNA sequencing and as site-specific mutagens. This and related activity has created an increased need for the chemical synthesis of oligonucleotides and small genes. The present invention greatly simplifies and increases the efficiency of the task of preparing synthetic oligonucleotides.
2. Description of the Prior Art
In the field of peptide chemistry, solid phase peptide synthesis ("SPPS") was introduced by Dr. R. Bruce Merrifield in 1963 when Dr. Merrifield attached a growing peptide chain to a solid support. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154. The procedures enunciated by Dr. Merrifield for SPPS were as follows: An amino acid corresponding to the C-terminal of the target peptide is covalently attached to an insoluble polymeric support ("the resin"). The next amino acid, with a protected .alpha.-amino acid, is activated and reacted with the resin-bound amino acid to yield an amino-protected dipeptide on the resin. Excess reactants and co-products are removed by filtration and washing. The amino-protecting group is removed by and chain extension is continued with the third and subsequent protected amino acids. After the target protected peptide chain has been built up in this stepwise fashion, all side chain groups are removed and the anchoring bond between the peptide and the resin is cleaved by suitable chemical means thereby releasing the crude peptide product into solution. The desired peptide then undergoes an extensive purification procedure and is then characterized. Kent, S. & Clark-Lewis, I., "Modern Methods for the Chemical Synthesis of Biologically Active Peptides," Division of Biology 147-75, California Institute of Technology, Pasadena, Calif. 91125 U.S.A.; Houghten, R. A., Chang, W. C. & Li, C. H. (1980), Int. J. Pept. Protein Res., 16, 311-320; Houghten, R A., Ostresh, J. M. & Klipstein, F. A. (1984), Eur. J. Biochem., 145, 157-162; Stewart, J. M. & Young, J. D., Solid Phase Peptide Synthesis, Pierce Chemical Company (2d ed. 1984). See, also, Geysen, H. M., 20 Meloen, R. H. & Baretling, S. J. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002; Matthes, H. W. D., Zenke, W. M., Grundstrom, T. Staub, A., Wintzerith, M. & Chambon P., (1984) The EMBO Journal, 3, 801-805.
The resin employed in standard SPPS is known as the "Merrifield resin" and is a polystyrene bead of 100-200 microns in size. The resin typically contains 0.5-2.0% divinylbenzene cross-linkage and contains 0.2 to 0.8 mmole of p-chloromethyl groups per gram resin. The number of p-chloromethyl groups determines the number of individual chains per gram and their ultimate size. The size of the bead allows for a rapid penetration of reagents in SPPS. The percentage of cross-linkage determines the extent to which the resin shrinks and swells during solvent changes. A large shrink-and-swell effect is preferred.
In the field of oligonucleotide chemistry, on the other hand, Dr. Khorana has developed techniques, which were used by others in solution-phase synthesis, for solid-phase synthesis. In doing so, Dr. Khorana eliminated the intensive purification procedures required between each chemical step; the solid-phase procedure only required filtration and rinsing of the solid support with fresh solvent. Solid-phase synthesis permitted chemists to add 15-16 nucleotides per day rather than four or five nucleotides per week.
While the solid phase techniques had revolutionized biomedical research in industry and academia, this procedure has remained essentially unchanged since its inception in the early 1960's. With the explosive pace at which biotechnical research has been advancing in the industrialized nations of the world, substantially more oligonucleotides, particularly analogs, of greater complexity are needed in industry and research than ever before. The ever increasing demand for analog oligonucleotides has been approached in several ways, but no approach thus far, has proven completely satisfactory.